Regulation of Herpes Simplex Type 1 Infection in Corneal Neurons

描述（由申请人提供）：1型单纯疱疹病毒（HSV）是感染性角膜失明的主要原因。它通过角膜神经中潜伏的病毒库的再激活引起眼病。尽管进行了广泛的研究，但调节HSV感染神经元的机制尚未得到很好的表征。更好地理解这一点对于确定新的治疗策略至关重要。我们开发了一种新的培养系统，利用分离的成年小鼠三叉神经节（TG）来研究HSV在神经元中的感染。我们实验室的初步数据表明，与它在复制细胞中的作用不同，病毒即时早期（IE）基因产物ICP27限制了神经元（尤其是A5+神经节神经元）的多产性病毒感染，并促进了病毒潜伏期。在目前的提案中，我们将进一步表征ICP27的这种新功能，并研究ICP27实现这些功能的机制。在本提案的前两个具体目标中，我们将利用ICP27零突变体、表达动力学延迟的ICP27启动子突变体和不同ICP27功能域缺失的病毒突变体来表征ICP27在限制生产感染和促进病毒潜伏期方面的作用。我们将进一步表征ICP27在限制神经元感染中的作用，通过使用新的AAV载体来有效地转导ICP27的感觉神经元。我们的初步数据还表明，复制细胞中的晚期病毒蛋白VP16在TG神经元中表达得非常早，并且ICP27限制了VP16和ICP4在培养的TG神经元中的转录。在第三个特定目标中，我们将进一步表征ICP27介导的ICP4和VP16转录抑制，并测试关于实现这种抑制方式的假设。最后，我们将验证ICP27限制A5+生产感染的假设

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) type 1 is a leading cause of infectious corneal blindness. It causes eye disease by reactivation from a latent viral reservoi in corneal nerves. Despite extensive research, the mechanisms that regulate HSV infection in neurons are not well characterized. A better understanding of this is critical for identifying new therapeutic strategies. We have developed a novel culture system, using dissociated adult murine trigeminal ganglia (TG), for studying HSV infection in neurons. Preliminary data from our lab indicates that, unlike its role in replicating cells, the viral immediate early (IE) gene produt, ICP27, restricts productive viral infection in neurons, especially in A5+ ganglionic neurons, and promotes viral latency. In the current proposal we will further characterize this novel function fo ICP27, as well as study the mechanisms by which ICP27 accomplishes these functions. In the first two specific aims of this proposal, we will characterize the role that ICP27 plays in restricting productive infection and promoting viral latency, using ICP27 null mutants, an ICP27 promoter mutant with delayed kinetics of expression, and viral mutants with deletions in different ICP27 functional domains. We will further characterize the role of ICP27 in restricting infection i neurons through the use of novel AAV vectors for the efficient transduction of sensory neurons with ICP27. Our preliminary data also suggests that VP16, a late viral protein in replicating cells is expressed very early in TG neurons, and that ICP27 restricts transcription of both VP16 and ICP4 in cultured TG neurons. In the third specific aim we will further characterize ICP27 mediated inhibition of ICP4 and VP16 transcription and test hypotheses about the way in which this is achieved. Finally, we will test hypotheses that ICP27 restricts productive infection in A5+ neurons, in part, by restricting ICP4, VP16 and HCF1 to the cytoplasm, thus preventing transactivation of viral IE genes. These concepts and studies are innovative, and are a result of being able to directly study HSV infection in neurons, as well as being able to differentiate the outcome of infection in A5+ neurons, the major site of HSV latency. The outcome of these studies should generate new insights into the mechanisms regulating HSV infection of neurons; the first step in developing new therapy strategies.